The emergent properties of life require the dynamic interactions of macromolecules, the two major classes of which are proteins and nucleic acids. These interactions form macromolecular machines and dynamic liaisons that shape the cell, transmit information and control cellular behaviors, and pathogenic alterations in molecular interaction networks (interactomes) underlie disease. Our understanding and modulation of biological systems, as well as their pathologies, thus relies on the ability to elucidate and interpret these interactions and their dynamics. For nucleic acids, recent advances have led to an explosion of genomic data. However, proteins are incredibly diverse in their abundance and their properties, making them highly versatile for their dynamic tasks, but at the same time exceptionally difficult to analyze. It is for these reasons that the interactomic revolution still ags very far behind the genomic revolution. The proposed National Center for Dynamic Interactome Research (NCDIR) couples cell biology laboratories, an established mass spectrometry resource, a systems biology resource, and a computational structural biology center. The goal of the NCDIR is to synergistically pioneer new and improved approaches, integrating these technologies into a fundamentally novel pipeline approach to address the urgent need of the biomedical community for technologies that can rapidly, reliably and routinely identify and characterize the dynamic cellular interactome. We will begin by developing technologies for purifying and preserving, with high fidelity, various defined forms of the hierarchical arrangement of interactors surrounding any chosen macromolecule. We will then provide comprehensive, highly quantitative, detailed temporal and structural data for dynamic complexes. Such data will be used to generate structural and mechanistic models that are predictive, testable, actionable, and guide experiments to focus on those that are most informative. The models aim to provide the biomedical community with the means for rational target-based intervention and drug design strategies. These approaches will be developed, refined and beta-tested via a selected set of Driving Biological Projects and Collaborations that can enter and exit our pipeline at any point, and which present specific technological challenges. Critical to the design of the NCDIR is an effective training and dissemination program that is responsive to the urgent needs of the biomedical community.